A peptide is a compound composed of a plurality of amino acids condensed with one another through a peptide bond, and has various properties based on the amino acids as constituent components, such as hydrophilicity, hydrophobicity, acidity and basicity. Further, a peptide has an intrinsic conformation depending on an amino acid sequence. Because of these characteristics, a peptide has various functions such as expression of a bioactivity via an interaction with a protein or the like.
For example, a peptide compound having a bioactivity can be developed into a pharmaceutical product. At present, a large number of peptide pharmaceutical products have been approved and sold on the market. Therefore, an expectation of development of simple, efficient and widely used synthesis method of a peptide compound has been increased.
A peptide is synthesized by repeating a dehydration-condensation reaction between an amino group of one amino acid component and a carboxyl group of another amino acid component, i.e. a peptide chain elongation reaction, depending on the amino acid sequence of the peptide. Of the two amino acid components involved in this peptide bond formation, the component that provides the amino group is referred to as an amine component, and the component that provides the carboxyl group is referred to as an acid component.
In a chemical peptide synthesis method, an acid component is activated by any of various active ester methods or with a coupling reagent typified by dicyclohexylcarbodiimide (DCC), propanephosphonic anhydride (T3P) or the like, and is reacted with an amine component, thereby a peptide bond is formed. In the chemical synthesis method, a functional group which should not participate in a condensation reaction, such as a carboxyl group of an amine component or an amino group of an acid component, is protected; and a condensation reaction is carried out; and then, the protecting group for the N-terminal amino group of the resulting condensed product is removed to form a new amine component; so that the reaction is controlled in order to obtain a peptide having a desired amino acid sequence. In this chemical peptide synthesis method, there are mainly a solid phase method in which a peptide chain is elongated on a solid phase support and a liquid phase method in which a reaction is carried out in a liquid phase.
In the solid phase method, an amine component of which the C-terminal carboxyl group is protected in such a form that the C-terminal carboxyl group is bound to an insoluble solid phase support composed of, for example polystyrene, via a linker is used; a successive elongation reaction is carried out while the C-terminal carboxyl group is bound to the solid phase support; the C-terminal carboxyl group is cleaved from the solid phase support after a desired sequence is completed to obtain a target peptide. The full automation of the solid phase method is easy to be achieved, since various peptides can be synthesized almost independent of their sequences by the method.
However, it is generally difficult in the solid phase method to completely react all amine components on the solid phase support, since an inhomogeneous reaction of a solid-liquid two phase system is carried out and the reaction site of the amine component is sterically crowded on the solid phase support. When a part of amine components bound to the solid phase support remain in an unreacted state in the condensation reaction, it is necessary to completely react the amine components using a large excess of reagents such as an acid component and a coupling reagent.
Further, it is difficult in the solid phase method to monitor a reaction conversion ratio or a quality. In addition, the impure peptide cannot be removed at all until the desired sequence is completed and cleaved from the solid phase support, since the by-produced impure peptide is bound to the solid phase support together with the target peptide. It is generally difficult to remove the impure peptide, since the impure peptide has a similar property to that of the target peptide due to a common partial structure among the peptides. From the above reasons, the solid phase method is incomplete particularly as a method of synthesizing a peptide compound of high purity.
Furthermore, when the solid phase method is applied to the production of a peptide compound on an industrial scale, there is a restriction in terms of the production facility and the like; and a relatively expensive solid phase support and a large amount of reagents and a solvent are used; and also a large amount of waste is generated in proportion to the amount of the used reagent or solvent; therefore, a high cost is required for a raw material and waste liquid disposal. Accordingly, it is hard to say that the solid phase method is an economically advantageous method.
On the other hand, the liquid phase method is a method in which a condensation reaction for forming a peptide bond and a deprotection reaction for removing a protecting group for the N-terminal amino group of the resulting condensed product to form a new amine component are carried out in a liquid phase (solution) by using a protecting group capable of making the resulting condensed product soluble in a reaction solvent as a protecting group for a carboxyl group of an amine component. In the liquid phase method, an impure peptide can be removed by purification in the middle stage of the peptide chain elongation. However, when a side chain functional group of an amino acid is not protected, complicated purification procedures such as chromatography purification of an intermediate, a crystallization procedure and a crystal washing procedure with a plurality of solvent systems are required for the respective condensation steps (refer to Non-patent document 1). Further, even when an amino acid of which a side chain functional group is completely protected is used, the method has a disadvantage that an impure peptide is by-produced and the purity of the target peptide is decreased, since an active ester remaining in a solution cannot be completely decomposed (refer to Non-patent document 2).
From the above circumstances, development of a liquid phase peptide synthesis method in which purification procedures for an intermediate are omitted as much as possible and a target peptide with high purity can be obtained by a simple procedure is demanded. Recently, a continuous liquid phase peptide synthesis method in which the respective procedures for peptide synthesis are simplified while maintaining the purity of the target peptide high has been developed.
As one of the continuous synthesis methods, there is the following liquid phase synthesis method reported by Carpino et al. (refer to Non-patent document 3 and Patent document 1). In the method, after a peptide condensation reaction using an excess amount of an acid component of which the N-terminal amino group is protected by a 9-fluorenylmethyloxycarbonyl (Fmoc) group and an amine component of which the C-terminal carboxyl group is protected by a t-butyl group, an active ester remaining in the solution is converted to an amide form to make the active ester harmless using a scavenger such as tris-(2-aminoethyl)amine; and at the same time, the protecting group for the amino group of the active ester is removed. Further, the removal of the protecting group for the N-terminal amino group of the target condensed product is also allowed to proceed, and decomposition of the active ester and removal of the protecting group for the N-terminal amino group are completed at the same time by using the above-mentioned scavenger. A compound generated by this active ester decomposition reaction is easily removed into an aqueous layer by washing with a weakly acidic aqueous solution. By repeating said series of procedures, a target peptide compound of high purity can be continuously synthesized.
Further, in the DioRaSSP method by Diosynth (refer to Patent document 2) which is a similar continuous synthesis method, after condensation using an excess amount of an acid component of which the N-terminal amino group is protected by a benzyloxycarbonyl (Z) group and an amine component of which the C-terminal carboxyl group is protected by a t-butyl group, an active ester remaining in the solution is converted to an amide form to make the active ester harmless using β-alanine benzyl ester as a scavenger. The amide form generated by the active ester decomposition reaction is converted to a decomposed product with a high water solubility by removing both protecting groups at the N-terminus and the C-terminus in the deprotection reaction of the N-terminal amino group of the condensed product through the subsequent catalytic hydrogenation; therefore, the resulting decomposed product is easily removed into an aqueous layer by a washing procedure. By repeating said series of procedures, a target peptide of high purity can be continuously synthesized.
These methods are capable of obtaining a target peptide compound with relatively high purity while simplifying the complicated purification procedures in the respective condensation steps, which were the problems in the conventional liquid phase synthesis method. Further, the continuous liquid phase synthesis method can be said to have high usefulness in the chemical peptide synthesis.
However, a protecting group for a target peptide is restricted in both of the method by Carpino et al. and the DioRaSSP method by the type of a scavenger for decomposing an active ester. In addition, it is necessary to use an expensive amine or unnatural amino acid derivative as the scavenger. Accordingly, development of a method of decomposing an active ester, which can be more widely used and is excellent in economical efficiency without depending on the type of a protected amino acid to be used and further without using an expensive reagent, has been demanded.
It is ideal in the liquid phase method that a target peptide compound and intermediate peptide thereof are dissolved in a liquid phase, and it is preferred that at least such peptides are homogenously dispersed in a liquid phase medium. Even when an organic solvent solution of the peptides is in a state of an emulsion or a gel, a problem does not arise in a reaction and a post-treatment if the peptides are in a homogenously dispersed state in the liquid phase medium. On the other hand, when the peptide compounds are aggregated and formed into an aggregate or the like, a problem arises that an unreacted amine component is incorporated into the aggregate and the reaction is not completed or a liquid separation procedure at the time of a post-treatment cannot be carried out. Accordingly, it has been demanded in the liquid phase method to develop a widely used novel medium (solvent system) capable of homogenously dispersing a wide range of peptide compounds in a liquid phase medium independent of their amino acid sequences.
As described above, a widely used chemical synthesis method capable of efficiently synthesizing a peptide compound with high purity having a desired amino acid sequence has not been fully established. Accordingly, development of a method of efficiently decomposing an active ester which greatly affect the quality of a peptide and a solvent system with a high solubility for peptides which are suitable for the respective procedures such as liquid separation in a continuous liquid phase method have been particularly and strongly demanded.    Patent document 1: U.S. Pat. No. 5,516,891    Patent document 2: JP-A-2003-55396    Non-patent document 1: Izumiya et al., “Pepuchido Gosei no Kiso to Jikken” (Basic Concepts and Experiments of Peptide Synthesis), Maruzen Co., Ltd. (1985)    Non-patent document 2: Bull. Chem. Soc. Jpn., 55, 2165 (1982)    Non-patent document 3: Org. Proc. Res. Dev., 7, 28 (2003)